The human epidermal growth factor (EGF) family of tyrosine kinase receptors consists of ErbB1 (EGFR), ErbB2 (Neu), ErbB3, and ErbB4, and plays key roles in cancer progression. Dysregulation of ErbB signaling due to hyperactivation of receptors (such as mutated EGFR or ErbB2 overexpression), overexpression of ligands (such as TGF-? or heregulins), or augmented downstream signaling (such as enhanced PI3K signaling) plays a major role in the etiology of breast cancer. We found that ligand stimulation of ErbB1 and ErbB3 receptors leads to a strong activation of the small GTPase Rac1 to promote mitogenesis and motility. The search for Rac-GEFs that mediate Rac1 activation by ErbB ligands in breast cancer cells revealed a prominent role for phosphatidylinositol-3,4,5-triphosphate-dependent Rac exchanger-1 (P-Rex1), a Rac-GEF originally identified in neutrophils but not studied in any cancer so far. Strikingly, P-Rex1 is highly expressed in most breast cancer cell lines as well as in tumors. P-Rex1 is a PI3K- and G??-dependent Rac-GEF, and coincidentally, ErbB receptor-mediated activation of Rac1 is inhibited by Pertusis toxin, which prevents G23 subunit release from heterotrimeric Gi proteins, by the PI3K inhibitor wortmannin, and by inhibition/depletion of PI3K?, a class Ib PI3K that is activated by G?? subunits. Moreover, ErbB3-induced activation of Rac1 is dependent on the transactivation of CXCR4, a Gi-coupled receptor widely implicated in breast cancer progression, and Rac1 activation by direct stimulation of CXCR4 with the chemokine SDF-11 also depends on P-Rex1. Therefore, P-Rex1 is a common mediator of Rac activation by ErbB receptors and GPCRs in breast cancer cells. In Specific Aim 1 we will investigate whether ligand stimulation of ErbB receptors can promote the activation of P-Rex1 in breast cancer cells, and assess the functional consequences for such activation, including actin cytoskeleton reorganization, migration, and proliferation. Translocation studies as well as FRET approaches will be used to address the relative contribution of discrete PI3K isozymes and the CXCR4-G?? pathway to P-Rex1 and Rac1 activation. In Specific Aim 2 we will determine whether RNAi depletion of P-Rex1 affects tumorigenicity and metastatic dissemination of breast cancer cells driven by overexpression of an ErbB3 ligand. We will also assess the involvement of P-Rex1 in anti-estrogen resistance. In Specific Aim 3 we will determine if Rac signaling via P-Rex1 contributes to ErbB2-driven tumorigenesis and metastasis, and whether this is mediated by the CXCR4-G??-PI3K? pathway. We will generate mammary-specific mouse models for P-Rex1 overexpression and determine whether it can lead to tumor formation or cooperate with ErbB2 overexpression. In Specific Aim 4 the goal is to determine whether P-Rex1 is a hallmark of human breast cancer, and P-Rex1 expression in human tumors correlates with signaling parameters. The identification of P-Rex1 as an effector of ErbB receptors may have major relevance for understanding the molecular basis and etiology of breast cancer as well as significant prognostic and therapeutic implications.